Ionic liquid-assisted SDS-PAGE to improve human serum protein separation

Research Article

Ionic liquid-assisted SDS-PAGE to improvehuman serum protein separation

Ionic liquid (IL)-assisted sodium dodecyl sulfate polyacrylamide gel electrophoresis

(ILs-SDS-PAGE) was presented to improve protein separation. ILs were employed during

the preparation process of polyacrylamide gel, then the modified gel was used for

commercial protein marker, binary bovine serum albumin/lysozyme (BSA/Lyz) and

human serum separation. The influence of ionic liquid concentration, cation alkyl chain

length, cation and anion types on proteins separation were investigated. The results

showed that ILs played a role in improving some protein separation, and ILs-SDS-PAGE

provided higher resolution and separation efficiency than ordinary SDS-PAGE for low

and middle relative molecular mass proteins in human serum. In addition, the principle

of ILs-SDS-PAGE was discussed and the comparison of ILs-SDS-PAGE with ordinary

SDS-PAGE and Native PAGE was made.

Keywords:

Human serum / ILs-SDS-PAGE / Ionic liquids / Protein separationDOI 10.1002/elps.201100184

1 Introduction

Since sequencing of the complete human genome was

achieved in 2003, proteomics, which has been expansively

applied in many fields such as basic bioscience research,

clinical diagnosis, biomarker discovery and therapeutic

applications, has become one of the central topics and is

making tremendous progress nowadays [1, 2].

Progress of proteomics is strongly dependent on the

development of protein separation techniques and MS tech-

nology [3]. The ordinary gel-based electrophoresis techniques,

SDS-PAGE [4–7] and 2-DE [5, 6, 8–10] as well as Native PAGE

(N-PAGE) [11, 12] and Blue-native PAGE (BN-PAGE) [9, 13, 14],

are important and universally used methods for protein

separation. SDS-PAGE, one of the basic and powerful methods

with a long history [15–17], is also the fundamental of ordinary

2-DE, which is developed based on IEF and SDS-PAGE [9, 18].

N-PAGE and BN-PAGE, different from all SDS-PAGE-based

electrophoresis, are capable of separating native and catalytic

active membrane proteins, which avoid the denaturation of

proteins with SDS [13, 14]. Currently, 1-D gel is more often

used for proteomic analysis [3]. The principle of SDS-PAGE is

that electrophoretic mobility of protein is relevant only to its Mr

since the use of SDS and b-mercaptoethanol (BME) or DTT,

which denatures original proteins and eliminates protein’s

original surface charge and form, then gives rise to Mr-based

SDS–protein complex [15]. The mobility of SDS–protein

complex in polyacrylamide gel (PAG) is determined by its Mr

and the permeability of gel. Therefore, SDS-PAGE limits high-

resolution separation of protein sample with similar proteins

Mr. So, the improvement of ordinary SDS-PAGE by integrating

other separation factors is very meaningful to complex protein

sample separation, which will also benefit other SDS-PAGE-

based electrophoreses.

Some powerful and elegant modified gel-based electro-

phoresis techniques have been successfully evolved to improve

protein separation in modified SDS-PAGE. Tricine-SDS-PAGE

is an important method for the separation of proteins in the

mass range 1–100 kDa [19]. Phosphate-affinity SDS-PAGE is

based on phosphate-affinity interactions by the modification

Tao Zhang1�

Qingqing Gai1�

Feng Qu1

Yukui Zhang2��

1School of Life Science, BeijingInstitute of Technology, Beijing,P. R. China

2Dalian Institute of ChemicalPhysics, Chinese Academy ofSciences, Dalian, P. R. China

Received March 22, 2011Revised May 14, 2011Accepted May 16, 2011

Abbreviations: AAm, acrylamide; APS, ammoniumpersulphate; [C4mim]BF4, 1-butyl-3-methylimidazoliumtetrafluoroborate; [C2mim]BF4, 1-ethyl-3-methylimidazoliumtetrafluoroborate; [C4mim]Br, 1-butyl-3-methylimidazoliumbromide; [C4mpd]Br, 1-butyl-3-methylpyridinium bromide;

[C6mim]BF4, 1-hexyl-3-methylimidazolium tetrafluoroborate;

[C8mim]BF4, 1-octyl-3-methylimidazolium tetrafluoroborate;

[C4mprd]Br, 1-butyl-1-methylpyrrolidinium bromide; Gly,

Glycine; HZ, high Mr zone; ILs, ionic liquids; ILs-PAG, ILs-polyacrylamide gel; LZ, low Mr zone; MZ, moderate Mr zone;

N-PAGE, Native PAGE; PAG, polyacrylamide gel; RML/B, therelative mobility ratio of Lyz to BSA

�These authors contributed equally to this work.��Additional corresponding author: Professor Yukui Zhang

E-mail: [email protected]

Correspondence: Professor Feng Qu, School of Life Science,Beijing Institute of Technology, 5th South Zhongguancun Street,Haidian District, Beijing 100081, P. R. ChinaE-mail: [email protected]: 186-10-68918015

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2011, 32, 2904–29102904

of polyacrylamide gel using Phos-tag [20–22]. The use of

additives, such as antigen, metal ions, pigments, proteins,

sugars and lectin, can associate or interact with the protein

during capillary electrophoresis and shift protein’s electro-

phoretic mobility in order to get better separation [23]. These

methods are typically limited to protein separation based on

the specificity of affinity interaction, which need special affinity

ligand and lack universality. For conventional proteins elec-

trophoresis, therefore, the simple modification of SDS-PAGE

for complex protein sample separation will have great potential

for a wide application of protein analysis.

Ionic liquids (ILs) have aroused considerable interest in

bio-catalysis [24, 25] and protein separation [26, 27]. An

attractive feature of ILs is their structure designable proper-

ties of cationic and anionic components, which can be

applied to introduce chemical and biochemical functionality

and results in specific bio-catalysis and bio-separation effect

[28, 29]. Another feature of ILs is the inherent amphiphilicity

of cation, so that they may be considered as short-chain

cationic surfactants [30]. Therefore, the controllable structure

property and amphiphilicity of ILs play an important role in

separation, such as they have been used either as a functional

group fixed on stationary phase of HPLC [31] or as running

buffer additives in capillary electrophoresis [32–36] and

microchip [28, 37] for peptides or proteins separation.

In this work, the IL-assisted SDS-PAGE (ILs-SDS-

PAGE) was presented for the separation of commercial

protein marker and binary BSA/lysozyme solution (BSA/

Lyz). The performance of ILs-SDS-PAGE for human serum

was evaluated, which improved low and middle Mr serum

protein separation with higher resolution, and more protein

bands were observed comparing with original SDS-PAGE.

The influence of ILs cation and anion type and concentra-

tion on the separation was investigated. In addition, the

principle of IL-assisted SDS-PAGE was discussed and

the comparison among ILs-SDS-PAGE, SDS-PAGE and

N-PAGE was made. The modification of SDS-PAGE by ILs

provides the potential application and universality for some

gel-based protein separation. To our knowledge, there is no

report of ILs application in SDS-PAGE for standard and

human serum protein separation.

2 Materials and methods

2.1 Reagents and solutions

99.9% m/m purity of ILs of 1-ethyl-3-methylimidazolium

tetrafluoroborate ([C2mim]BF4), 1-butyl-3-methylimidazo-

lium tetrafluoroborate ([C4mim]BF4), 1-hexyl-3-methylimi-

dazolium tetrafluoroborate ([C6mim]BF4), 1-octyl-

3-methylimidazolium tetrafluoroborate ([C8mim]BF4),

1-butyl-3-methylimidazolium chloride ([C4mim]Cl), 1-butyl-

3-methylimidazolium bromide ([C4mim]Br), 1-butyl-3-

methylpyridinium bromide ([C4mpd]Br) and 1-butyl-3-

methylpyrrolidinium bromide ([C4mprd]Br) were purchased

from Chengjie Chemical (Shanghai, China).

TEMED, Bis, acrylamide (AAm), SDS and ammonium

persulphate (APS) were provided by Sigma-Aldrich (Tokyo,

Japan).

SDS-sample buffer (100 mM Tris-HCl, 200 mM DTT,

4% m/v SDS, 20% m/v glycerine, 0.1% m/v bromophenol

blue, pH 6.8) and protein molecular marker (band 1, Mr

14.4 kDa; band 2, Mr 20.0 kDa; band 3, Mr 26.0 kDa; band 4,

Mr 33.0 kDa; band 5, Mr 45.0 kDa; band 6, Mr 66.2 kDa;

band 7, Mr 94.0 kDa) were supplied by Tiangen Biotech

(Beijing, China).

BSA, Lyz, Tris base and CBB R250 were purchased from

Amresco (St. Louis, MO, USA). Glycine (Gly) was from

Biodee Biotech (Beijing, China). Human serum was

supplied from Ruite Biotech (Guangzhou, China). All other

reagents were analytical grade and all solutions were

prepared by double distilled water.

2.2 Apparatus

Mini-4 gel electrophoresis system (Kaiyuan, Beijing, China)

was used for protein electrophoresis separation.

2.3 Procedure of ILs-SDS-PAGE

Figure 1 shows the ILs-SDS-PAGE procedure, which

consisted of four steps: ILs-polyacrylamide gel (ILs-PAG)

preparation (gel formation); SDS–protein complex prepara-

tion; protein electrophoresis with SDS-running buffer;

staining, decolorization and analysis of protein. In ILs-

SDS-PAGE, the modification was made at the first step of

ILs-PAG gel formation, in which the use of SDS was

replaced by ILs.

2.4 Preparation of PAG stock solution

30% m/v AAm stock solution (29.25% T, 0.75% C, m/v) was

prepared by adding 29.25 g AAm and 0.75 g Bis in 100 mL of

double distilled water and stored at 41C, which was used as a

stock solution for the next ILs-PAG preparation.

2.5 Preparation of ILs-PAG

ILs-PAG was performed in a vertical discontinuous gel

system, which consisted of separating ILs-PAG and stacking

Figure 1. Schematic diagram of ILs-SDS-PAGE procedure.

Electrophoresis 2011, 32, 2904–2910 General 2905


ILs-PAG as well as the corresponding ILs concentration. ILs-

PAG with the dimensions of approx. 100� 75 mm

(width� length) and a thickness of 1.0 mm was made in

this work.

Separating resolving gel was prepared by mixing

2.00 mL of stock solution, 1.25 mL of Tris-HCl (1.5 M, pH

8.8), 1.75 mL of double distilled water. ILs-PAG formed

with 0.05–1.0% m/v ILs concentrations were achieved by

adding the desired volume of ILs into the above

resolving gel. After adding 0.005 mL of TEMED and

0.05 mL of APS, 5 mL separating ILs resolving gel was

transferred to the assembled glass plates and allowed to

polymerize for 50 min, then ILs were immobilized to form

separating ILs-PAG (11.7% T, 0.3% C, m/v) for protein

separation.

The stacking resolving gels were prepared by mixing

0.67 mL of AAm stock solution, 1.25 mL of Tris-HCl

(0.5 M, pH 6.8) and 3.08 mL of double distilled water.

ILs were added in the same way at the same concentration

as separating ILs-PAG, and then added 0.005 mL of

TEMED and 0.05 mL of APS into the gel. Comb was

inserted after stacking resolving gel was loaded and left for

3 h. Then, the comb was removed and the gel ILs-PAG

(3.9% T, 0.1% C, m/v) was formed, waiting for loading and

running.

2.6 Preparation of SDS–protein complex

The commercial protein marker can be loaded directly

without further treatment. About 10 mL 0.2 mg/mL

binary BSA/Lyz solution or 10 mL 10-fold diluted human

serum was mixed with 10 mL commercial SDS-sample

buffer. The mixtures were then boiled at 951C for

5 min, and subsequently cooled in the refrigerator at 41C,

which was used as a protein sample for the next

electrophoresis.

2.7 Protein electrophoresis

Optimized 10 mL sample [38] was loaded in the lanes of

stacking ILs-PAG respectively. The gels were then subjected

to electrophoresis at a constant voltage of 100 V with a SDS-

running buffer (50 mM Tris, 10 mM Gly, 10 mM SDS, pH

8.0) for 8 min. When the samples entered the separating

ILs-PAG, the voltage was turned to 140 V and kept constant

for 65 min.

2.8 Staining and decolorization of gel

At the end of electrophoresis, ILs-PAG was removed from

the glass plates and was washed with double distilled water

to remove SDS. The gel was stained with 0.1% m/v CCB

R-250 solution for 40 min with gentle shaking at 50 rpm.

Then, it was decolored in a mixture of 20% v/v methanol,

20% v/v acetic acid and double distilled water to remove the

background [39].

3 Results and discussion

3.1 Effect of alkyl chain length of ILs cation and

concentration on protein marker separation

ILs with C2–C8 alkyl chain ([C2mim]BF4, [C4mim]BF4,

[C6mim]BF4 and [C8mim]BF4) were used in the preparation

of gel, respectively. 0.05% m/v [C2mim]BF4 and [C4mim]BF4

caused seven clear protein bands (Fig. 2A), which mani-

fested the success of proteins marker separation with the

use of ILs. However, the migration distance of seven

proteins showed obvious difference. Comparing with in C2,

protein bands 1–4 in C4 exhibited apparent shorter

migration distance, which indicated that the migration of

low Mr marker protein was retarded by C4 ILs, and low Mr

protein migration could be adjusted with the aid of ILs. With

higher concentration 0.1–0.4% m/v, both [C2mim]BF4 and

[C4mim]BF4 gave clear separation (Fig. 2B–D), but 0.6–1.0%

m/v [C4mim]BF4 caused protein bands 1–3 obscure.

With longer alkyl chain, 0.05–1.0% m/v [C6mim]BF4

and [C8mim]BF4) damaged the separation of marker

proteins (Fig. 2A–F). The results indicated that 0.05% m/v

longer alkyl chains C6 and C8 in ILs cation would deterio-

rate protein separation seriously.

Above results showed that the use of ILs played a role in

modifying protein electrophoresis. At the same ILs concen-

tration, different alkyl chains caused apparent differences on

protein marker separation, which indicated that the protein

separation in ILs-SDS-PAGE was affected by the alkyl chain of

ILs cation. This may resulted from that the longer alkyl chain

cation provided stronger interaction with SDS, then damaged

the SDS–protein complex and also deteriorated protein

separation. Shorter alkyl chains C2 and C4 with optimized

concentration were suitable for protein marker separation. In

this experiment, lower concentration of ILs displayed a better

result (Fig. 2A–C comparing with Fig. 2D–F).

3.2 Comparison of ILs-SDS-PAGE and ordinary SDS-

PAGE

The migration distance of marker proteins separated by

0.05% m/v ILs-SDS-PAGE ([C2mim]BF4, [C4mim]BF4) and

ordinary SDS-PAGE was compared (Fig. 3). The results

showed that the mobility of seven marker proteins (bands

1–7) in [C2mim]BF4-SDS-PAGE were nearly equal to that in

SDS-PAGE. However, the longer alkyl chain C4 slowed

down the mobility distance of lower Mr proteins (bands 1–4)

obviously. Take band 1, for example, the mobility distance of

6.0 cm in SDS-PAGE decreased to 5.9 cm in [C2mim]BF4-

SDS-PAGE and then shortened to 5.2 cm in [C4mim]BF4-

SDS-PAGE. So, with the aid of [C4mim]BF4, the mobility

distance of low Mr marker proteins could be adjusted.

Electrophoresis 2011, 32, 2904–29102906 T. Zhang et al.


3.3 Effect of ILs alkyl chain length and concentration

on separation of Lyz and BSA

Lyz (14.3 kDa, pI 11.1) and BSA (67 kDa, pI 4.7) were

chosen as model proteins in ILs-SDS-PAGE due to their

apparent different Mr’s and pI’s. With [C2mim]BF4 and

[C4mim]BF4 concentration increased from 0.05 to 1.0% m/v,

the mobility distance of model proteins Lyz and BSA

decreased. In [C4mim]BF4-SDS-PAGE, the distance of Lyz

decreased from 5.2 to 5.0 cm, and BSA changed from 1.4 to

1.1 cm. Figure 4 shows the relative mobility ratio of Lyz to

BSA (RML/B) increased with ILs concentration increased.

Since 0.05% m/v [C6mim]BF4 and [C8mim]BF4 made Lyz

band disappear, the RML/B value could not be obtained.

Comparing with ordinary SDS-PAGE, 0.05–1.0% m/v

[C2mim]BF4 caused the increase in RML/B. However,

0.05–0.2% m/v [C4mim]BF4 caused the decrease in RML/B.

Then, when concentration increased from 0.4 to 1.0% m/v,

the increase in RML/B was observed. About 0.6% m/v

[C4mim]BF4-SDS-PAGE provided the highest RML/B 4.6.

Above results indicated the difference in RML/B value with

ILs concentration change, and the difference between RML/

B value in ILs-SDS-PAGE and in ordinary SDS-PAGE. Since

higher RML/B indicated the better separation selectivity and

higher resolution, with the optimized ILs type and concen-

tration, some proteins separation could be modified.

3.4 Application of ILs-SDS-PAGE in human serum

separation

The application of ILs-SDS-PAGE in human serum separa-

tion was evaluated, and the ordinary SDS-PAGE was used as

Figure 2. Electrophoresis ofprotein marker (band 1, Mr

14.4 kDa; band 2, Mr 20.0 kDa;band 3, Mr 26.0 kDa; band 4,Mr 33.0 kDa; band 5, Mr

45.0 kDa; band 6, Mr 66.2 kDa;band 7, Mr 94.0 kDa) with[C2–C8mim]BF4. ILs concentra-tion (m/v): (A) 0.05%; (B) 0.1%;(C) 0.2%; (D) 0.4%; (E) 0.6%;(F) 1.0%.

1 2 3 4 5 6 70

1

2

3

4

5

6

The

Mob

ility

Dis

tanc

e of

Pro

tein

(cm

)

Band

SDS-PAGE [C2mim]BF4-SDS-PAGE

[C4mim]BF4-SDS-PAGE

Figure 3. The mobility distance of proteins marker in 0.05% m/vILs-SDS-PAGE.



control for comparison. Three types of ILs with different

cations but same alkyl chain length ([C4mim]Br, [C4mpd]Br

and [C4mprd]Br) at concentration of 0.2% m/v (Fig. 5A),

0.6% m/v (Fig. 5B) and 1.0% m/v (Fig. 5C) were employed.

The bands of human serum could be divided into three

zones: high Mr zone (HZ), in which proteins migrated

slower than HSA, the highest abundant proteins in human

serum; moderate Mr zone (MZ), proteins migrated close but

faster than HSA; low Mr zone (LZ), the fastest migration

proteins zone.

The results showed that the using of 0.6% m/v and

1.0% m/v of ILs gave rise to significant better performance

than ordinary SDS-PAGE in MZ and LZ proteins. More

clear bands can be observed in ILs-SDS-PAGE than in SDS-

PAGE. And the higher concentration of 1.0% m/v ILs

gave a better performance than 0.6% m/v ILs. Meanwhile,

three ILs type showed a little different resolution to

MZ and LZ proteins. [C4mpd]Br was more powerful in

improving MZ proteins resolution than [C4mim]Br or

[C4mprd]Br. At least six clear bands were obtained in 1.0%

m/v [C4mpd]Br-SDS-PAGE, which was the optimal result of

MZ proteins separation. For proteins in LZ, three types ILs

gave similar results. Apparent three bands were observed,

more than one band only in SDS-PAGE. The difference of

mobility distance of three bands could be seen, which

should attribute to the different effects of ILs types.

However, for HZ, the effect of ILs could not be convinced in

comparison with SDS-PAGE. These results clearly showed

the different separations caused by [C4mim]Br, [C4mpd]Br

and [C4mprd]Br, which may be derived from the cations

difference in ILs in hydrophobicity and positive charge

distribution.

Comparing with SDS-PAGE, the appearance of new

bands observed in MZ and LZ regions convinces the effect

of ILs, and highlights the possibility and potential of ILs in

improving low and middle Mr proteins separation.

The role of cation in ILs was confirmed by changing

anions in ILs. When human serum was separated by

[C4mim]BF4-SDS-PAGE, [C4mim]Cl-SDS-PAGE and

[C4mim]Br-SDS-PAGE, respectively, there was no great

difference in the three regions of HZ, MZ and LZ (Fig. 6),

which mean that the anion in ILs did not affect the human

serum protein separation. So, cation part in ILs dominated

ILs-PAG formation and the protein separation.

3.5 The separation principle of ILs-SDS-PAGE

Comparing with ordinary SDS-PAGE procedure, the only

difference of ILs-SDS-PAGE was in the formation of ILs-

PAG. During the preparation of PAG, the use of SDS was

replaced by ILs, then the same ordinary SDS-PAGE

procedure was followed (refer to Fig. 1). Like proteins in

SDS-PAGE, after proteins were treated with SDS-sample

buffer, proteins in ILs-SDS-PAGE were coated with SDS,

yielding the uniform negative charge. BME or DTT in SDS-

sample buffer destroyed the disulfide bond between the

amine acids in protein and eliminated the protein’s original

form difference. So all proteins would have roughly the

same mass-to-charge ratio, and existed in the form of

SDS–protein complex [15, 38, 40].

0.0 0.2 0.4 0.6 0.8 1.03.0

3.5

4.0

4.5

5.0

5.5

Rel

ativ

e M

obili

ty o

f L

yz/B

SA

Concentration of ILs in gel(% w/v)

[C2mim]BF

4 -SDS-PAGE

[C4mim]BF

4 -SDS-PAGE

SDS-PAGE

Figure 4. Relative mobility ratio of Lyz and BSA (RML/B) in ILs-SDS-PAGE and SDS-PAGE.

Figure 5. Comparison of SDS-PAGE and ILs-SDS-PAGE forhuman serum separation. ILsconcentration (m/v): (A) 0.2%;(B) 0.6%; (C) 1.0%.



The formation of ILs-PAG was based on the reaction of

AAm and Bis, cation in ILs could be embedded into the gel,

and the positive charge and hydrophobic alkyl chain in ILs

cation could provide some electrostatic and hydrophobic

interactions with the passing SDS–protein complex, which

played an important role in changing their electrophoretic

mobility.

Longer hydrophobic alkyl chain cation, as a surfactant,

could interact with SDS and neutralize the effect of

SDS, then damage the SDS–protein complex. So, ILs cation

with longer alkyl chain and higher concentration

would disturb the stable SDS–protein complex and also

deteriorate protein separation. Moreover, with the same

alkyl chain, different types of cation of imidazolium, pyri-

dinium and pyrrolidinium based also displayed a minor

difference.

In addition, when ILs were added only in running

buffer as an additive instead of adding in the process of gel

formation, the worse performance for human serum

protein separation was obtained (comparing lines 2 and 3 in

Fig. 7), which was attributed to the damage of SDS–protein

complex due to ILs cation existed in running buffer, which

also indicated that the use of ILs in gel formation was

necessary.

In ILs-SDS-PAGE, protein separation depended not

only on the permeability of ILs-PAG and protein Mr, but

also on the interaction between SDS–protein complex and

ILs cation, the use of ILs in ordinary SDS-PAGE benefited

the separation of SDS–protein complex with similar LZ and

MZ proteins, and improved their resolution to some extent.

The features of ILs-SDS-PAGE comparing with the most

widely used ordinary SDS-PAGE and N-PAGE are

summarized in Table 1.

4 Concluding remarks

The IL-assisted SDS-PAGE method is established by using ILs

as a substitute of SDS in gel formation. It improves the LZ and

Figure 7. Human serum electrophoresis. Line 1, SDS-PAGE; line2, SDS-PAGE with 1.0% m/v [C4mpd]Br in gel formation; line 3,SDS-PAGE with 1.0% m/v [C4mpd]Br only in running buffer.

Figure 6. Human serum electrophoresis in 0.60% m/v ILs.

Table 1. Comparison of ILs-SDS-PAGE with ordinary SDS-PAGE and N-PAGE

Methods ILs-SDS-PAGE SDS-PAGE [15–17] N-PAGE [11, 12]

Separation principle Mr and interaction between ILs

and protein

Mr Net charge, size and form

Protein sample Denaturation Denaturation Native

Use of SDS In sample buffer and running

buffer

In sample buffer, running buffer and

gel formation

None

Use of ILs In gel formation None None

Operating temperature Not required Not required 0–41C

Mr identification Direct Direct Method dependent

Target proteins All proteins simultaneously

detection

All proteins simultaneously detection Acidic or basic proteins in

one run

Resolution Higher Higher Lower

Major application Separation and identification Separation and identification Separation, identification and

activity analysis



MZ proteins separation in human serum, and provides higher

separation resolution and efficiency in comparison with

ordinary SDS-PAGE. Even though the similar effect might

be achieved by using gradient SDS-PAGE in some extent, the

presented ILs-SDS-PAGE process is obviously more conveni-

ent and simple for some complex sample, like serum.

The cation in ILs plays the important role for the

modification of protein separation in SDS-PAGE. Since ILs

have the characteristics of diversity and designable property,

the different separation performances of ILs highlights the

possibility of designing ILs or other molecular for the target

protein separation. Furthermore, since gel-based electro-

phoresis is a conventional method, IL-assisted SDS-PAGE

has the potential to become a powerful and universal tool for

protein separation and analysis.

The authors are grateful to the National Basic ResearchProgram of China (973 Program, No. 2007CB914101), theNational Nature Science Foundation of China (No. 20875009),the Academic Newcomer Project for Doctoral Candidates ofMinistry of Education of China, the Nursery Fund forOutstanding Doctoral Dissertation and the Special Science andTechnology Innovation Project for Postgraduate in BeijingInstitute of Technology for financial support.

The authors have declared no conflict of interest.

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Ionic liquid-assisted SDS-PAGE to improve human serum protein separation

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